The eye forms from the interaction of the optic vesicle, an out pocketing of the ventral forebrain, with the surface ectoderm of the head. Contact between these tissues leads to the formation of the lens placode, the first step in lens formation. Lens placode formation is required for the subsequent invagination of the optic vesicle to form the optic cup. Failure of these interactions leads to anophthalmia, microphthalmia or coloboma, which cause blindness or severely impaired vision. Despite their central importance in eye development, surprisingly little is known about the interactions between the optic vesicle and the lens-forming ectoderm. BMP4, probably produced by the optic vesicle and BMP receptors in the ectoderm are required for lens formation. Our recent data suggest that BMP signaling and Pax6, a transcription factor required to make the lens, function together in lens placode cells to promote lens formation. One aim of this study is to identify the mechanism(s) that coordinate the function of these two pathways. Two promising possibilities will be tested. We will determine whether BMP signaling in the ectoderm activates Pax6 by coordinae action of Smad proteins and the MAP kinases, Tak1 and p38, since p38 kinases are known to phosphorylate and activate Pax6. Homeodomain-interacting protein kinases (HIPKs) also phosphorylate and activate Pax6 and deletion of Hipk1 and 2 inhibits lens formation. We will determine whether BMP signaling activates the HIPKs to enhance Pax6 transcriptional activity and lens induction. Deletion of Pax6 in the ectoderm greatly reduced the extracellular matrix (ECM) between the ectoderm and the optic vesicle. This observation is consistent with an often overlooked hypothesis about the mechanism of lens placode formation, which we call the restricted expansion hypothesis. We showed in a recent paper that this hypothesis could explain lens placode formation. In the process of these experiments, we observed that lens placode formation is accompanied by thickening of the distal optic vesicle to form what we call the retinal placode. Disruption of lens placode formation prevented the formation of the retinal placode and the invagination to form the optic cup. Based on these observations, we formulated a simple model that explains retinal placode formation and its invagination to form the optic cup. We will test this model to determine if it explains the coordination of lens placode and optic cup formation. Our studies of optic cup morphogenesis are being conducted in collaboration with Dr. Larry Taber in our Biomedical Engineering Department. Dr. Taber's group is using time-lapse micro-OCT images to create a finite element model of optic cup invagination. We will test this model using mutants in which optic cup formation fails, leading to anophthalmia, or in which the ventral optic cup does not fuse, causing coloboma formation. The experiments proposed in this application will identify and test the mechanisms underlying the formation of the lens and optic cup that are required to produce a functional eye. PUBLIC HEALTH RELEVANCE: Failure of eye formation (anophthalmia), formation of a small, poorly-functioning eye (microphthalmia) and failure of the optic cup to close (coloboma) cause blindness or severe visual disability. Together, these defects are responsible for a substantial proportion of all childhood blindness. All three conditions are directly or indirectly associated with defects in communication between the two tissues that form the eye early in development, the optic vesicle and the surface ectoderm. The optic vesicle forms the retina and the surface ectoderm forms the lens. Our studies focus on how the lens-forming ectoderm responds to signals from the optic vesicle and how the surface ectoderm controls the formation of the optic cup and retina. To accomplish these aims, we will determine how signaling by BMP growth factors is coordinated with the function of Pax6, a transcription factor that is required for lens formation. We will also test a prediction that arose from our recent studies: that the extracellula matrix between the optic vesicle and the ectoderm is required for both lens and optic cup formation. We will also use an advanced imaging technique to generate and analyze a computer model of optic cup formation in chicken and mouse embryos. Using this approach, we will also test how two growth factors control the shape of the optic cup to permit it to function correctly and prevent coloboma formation. We expect these studies to provide a comprehensive analysis of the tissue interactions in early eye development and to reveal the critical events required for eye formation, a first step in preventing anophthalmia, microphthalmia and coloboma.